Hemolysis, damage to the structure of erythrocytes (red blood cells) resulting in a loss of hemoglobin, is generally undesired. Hemolysis can occur in vivo as the result of certain disease states or upon administration of certain therapeutic agents. In vitro, hemolysis can be induced during the handling and storage of blood, blood components or solutions, tissues or organs containing erythrocytes. Various chemical or pharmaceutical agents, as well as materials such as silica, clay, asbestos etc. can each cause hemolysis upon contact with erythrocytes. Because of their hemolytic properties, the use of many compounds and pharmaceutical agents that may have desirable pharmacologic properties may be limited in dosage or prevented from use altogether. For example, phenothiazine compounds are known to induce hemolysis above concentrations of the order of 0.1 mM. Chlorpromazine, a neuroleptic compound of the phenothiazine family exhibits hemolytic properties at concentrations in excess of 0.5 mM. Therefore the pharmaceutical use of chlorpromazine is restricted in dosage to avoid hemolytic side affects which would result at higher concentrations.
It has been previously reported that simultaneous use of 1.0 mM .beta.-cyclodextrin inhibits the hemolytic action of chloropromazine. (K. Uekama et al., Protective Effects of Cyclodextrins on Drug-Induced Hemolysis In Vitro, Effects of Cyclodextrins on Chlorpromazine Induced Hemolysis and Central Nervous System Responses, J. Pharm. Pharmacol., 33:707-710 (1981)). It was shown that protection from hemolytic effects of the drug can be obtained by virtue of the ability of cyclodextrin to include the offending molecules within the cavity of the cyclic, doughnut shaped cyclodextrin molecule. This internal complexing capability of cyclodextrins has been widely recognized (see for example W. Saenger, Angew. Chem. Int. Ed. Engl., 19. 344-362 1980) and has been applied for the delivery of thereby solubilized pharmaceutical compounds (see for example, K. Uekama, F. Hirayama, K. Esaki, M. Inoue, Chem. Pharm. Bull., 37, 76-70, 1979). By virtue of such complexing, the outside solution concentration of the included agent is reduced, and, for sufficient cyclodextrin present, can be reduced below the critical concentration effective in hemolysis.
Similar uses of cyclodextrin to inhibit hemolysis have been reported for other pharmaceutical agents. Examples are described in the following articles: Improvement of Local Irritation Induced with Intramuscular Injection of Tiamulin by Cyclodextrin Complexation, Y. Sato et al., Yakugazu Zasshi, 102:874-880 (1982); Protective Mechanism of .beta.-cyclodextrin for the Hemolysis Induced with Phenothiazine Neuroleptics In Vitro, T. Irie et al., J. Pharm. Dyn., 6:408-414 (1983); and Protective Effects of Cyclodextrin for the Local Irritation Induced by Aqueous Preparations of Flurbiprofen, K. Masuda et al., Yakugazu Zasshi, 104:1075-1082 (1982).
The use of .beta.-cyclodextrin to inhibit hemolysis, however, is limited. It is now well recognized that .beta.-cyclodextrin itself causes hemolysis at concentrations of about 2-3 mM and higher. Certain derivatives of .beta.-cyclodextrin induce hemolysis at even lower concentrations. For example, when hydroxyl groups are replaced with O-methyl groups, hemolysis is observed at levels below 1 mM .beta.-cyclodextrin. (T. Irie et al., Jr. Pharm. Dyn., 5:741-744 (1982)).
It is well known in the art (see the references to Saenger and Uekama noted above) that a molar quantity of cyclodextrin can include or host at most one but generally less than a molar quantity of pharmaceutical guest molecules. The quantity of pharmaceutical material that can be protected is therefore limited by the onset of hemolysis by the cyclodextrin concentration. Indeed, the beneficial effects of .beta.-cyclodextrin reported to inhibit hemolytic activity of pharmaceutical agents in the aforementioned references uniformly were based on concentrations of .beta.-cyclodextrin at or below 1.5 mM, and most frequently below 1.0 mM.
Aside from their use as hemolysis inhibitors, cyclodextrins, because of their unique inclusion capability and their water solubility, are useful for increasing the solubility of pharmaceutical agents of limited solubility by forming inclusion complexes with such agents. This application of cyclodextrin inclusion for solubilization of pharmaceutical or chemical agents of very low solubility is, of course, also limited in the amount of solubilization achievable, by the onset of hemolysis at the higher concentrations of cyclodextrins required, even though the agent itself is not hemolytically active.